Multistep reaction lateral flow capillary device

ABSTRACT

Disclosed is a lateral flow capillary device and uses thereof comprising a unipath bibulous capillary flow matrix and at least two reservoirs each in fluid communication with the capillary flow matrix wherein a reservoir contacts the capillary flow matrix through a passage having a rim pressing the matrix. The pressure that the rim applies on the matrix prevents leakage of liquids out of the capillary flow matrix at the reservoir/matrix interface, allowing accurate sequential draining of liquid from the reservoirs. During use of the disclosed lateral flow capillary device a static interface is formed between the first liquid and the second liquid in an interface creation zone inside the capillary flow matrix wherein the first amount and second amount are such that first liquid substantially remains in the first reservoir and the second liquid substantially remains in the second reservoir subsequent to the formation of the static interface and wherein the interface begins to move only subsequent to exhaustion of a liquid from a reservoir.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of application Ser. No. 11/883,329filed on Jul. 30, 2007, which is a National Phase of PCT PatentApplication No. PCT/IL2006/000121 having International Filing Date ofJan. 31, 2006, which claims the benefit of U.S. Provisional PatentApplication No. 60/647,774 filed on Jan. 31, 2005. The contents of theabove applications are all incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to the field of biology and detection ofanalytes from environmental and biological fluids, mainly atpoints-of-care more particularly, to an improved lateral flow capillarydevice and methods for using lateral flow capillary devices, for examplefor performing specific binding assays.

The use of specific binding assays is of great value in a variety ofclinical and other applications, see for example PCT patent applicationUS2004/031220 published as WO 2005/031355. Specific binding assaysinvolve the detection and preferably quantitative determination of ananalyte in a sample where the analyte is a member of a specific bindingpair consisting of a ligand and a receptor. The ligand and the receptorconstituting a specific binding pair are related in that the receptorand ligand specifically mutually bind. Specific binding assays includeimmunological assays involving reactions between antibodies andantigens, hybridization reactions of DNA and RNA, and other specificbinding reactions such as those involving hormone and other biologicalreceptors. Specific binding assays may be practiced according to avariety of methods known to the art. Such assays include competitivebinding assays, “direct” and “indirect” sandwich assays as described,for example, in U.S. Pat. No. 4,861,711; U.S. Pat. No. 5,120,643; U.S.Pat. No. 4,855,240 or EP 284,232.

Because the complex formed of by a specific binding reaction isgenerally not directly observable various techniques have been devisedfor labeling one member of the specific binding pair in order that thebinding reaction may be observed. Known labels include radiolabels,chromophores and fluorophores and enzymes the presence of which may bedetected by means of radiation detectors, spectrophotometers or thenaked eye. When a member of a specific binding pair is tagged with anenzyme label, a complex may be detected by the enzymatic activation of areaction system including a signal generating substrate/cofactor groupwherein a compound such as a dyestuff, is activated to produce adetectable signal.

Lateral flow capillary devices, such as lateral flow capillary device 10depicted in FIG. 1, are well known in the fields of analysis anddetection and are often used for quick and simple implementation ofspecific binding assay of analyte in a liquid sample 12. Sample 12 isplaced in lateral flow capillary device 10 through a reservoir 14 tocontact a liquid receiving zone 16 of a bibulous capillary flow matrix18. Receiving zone 16 includes a soluble labeled reagent configured tobind to the analyte which present in the sample 12. Sample 12 includingthe analyte bound to the labeled reagent, migrates by capillary flow tofill all of capillary flow matrix 18 and to migrate further into liquiddrain 23. During the capillary flow of sample 12 from liquid receivingzone 16 towards liquid drain 23, sample 12 passes reaction zone 20 whichis observable through an observation window 22. Reaction zone 20comprises an anti-analyte that together with the analyte constitutes aspecific binding pair. Analyte in sample 20 forms a complex with theanti analyte and is thus captured at reaction zone 20. As the labeledreagent is bound to the analyte, and as the analyte is concentrated atreaction zone 20, an observable signal is produced at the reaction zone20, where the intensity of the observable signal is related to theamount of analyte in the sample.

Lateral flow capillary devices such as device 10 are extremely useful asthese are simple to operate even by an unskilled person or undernon-laboratory conditions and are relatively cheap to produce.

One drawback of known lateral flow capillary devices such as device 10is that a sample evenly spreads in all directions until a border tocapillary flow is encountered, such as an edge of the capillary flowmatrix. Thus, sample and any analyte therein are distributed within theentire volume of the capillary flow matrix and wasted. It would beadvantageous to be able to enable transport of all of a sample added toa capillary flow matrix to the vicinity of a respective reaction zone.

An additional drawback of known lateral flow capillary devices is thatthese are not configured for multistep reactions. To perform a multistepbinding assay using a lateral flow capillarydevice such as device 10,reagent liquids are added serially. For example, a device 10 is providedwhere a liquid receiving zone 16 does not include a labeled reagent.

First, a sample 12 including analyte is added through reservoir 14,passes into capillary flow matrix 18 through liquid receiving zone 16and is transported by capillary flow to drain 23. When sample 12 passesthrough reaction zone 20, analyte in sample 20 forms a complex with theanti analyte located at reaction zone and is thus captured at reactionzone 20.

When all of sample 12 has drained into capillary flow matrix 18, a firstreagent liquid containing a labeled reagent configured to bind to theanalyte is added through reservoir 14, passes into capillary flow matrix18 through liquid receiving zone 16 and is transported by capillary flowto drain 23. When the first reagent liquid passes through reaction zone20, labeled reagent in the first reagent liquid binds to analytecaptured at the reaction zone.

When labeled reagent includes an enzyme, then when all of the firstreagent liquid has drained into capillary flow matrix 18, a secondreagent liquid containing an enzyme substrate is added through reservoir14, passes into capillary flow matrix 18 through liquid receiving zone16 and is transported by capillary flow to drain 23. When the secondreagent liquid passes through reaction zone 20, the enzyme substratetherein reacts with the enzyme label, producing a strong observablesignal at the reaction zone 20, where the intensity of the observablesignal is related to the amount of analyte in the sample.

It is known that multistep binding assays are significantly moresensitive and accurate than single step binding assays. Thus, there is adesire to perform multi step binding assays as described above. It isclear, however, that it is very difficult if not impossible to achieveaccurate and repeatable results for such a complex process without theuse of an expensive robotic system located in a laboratory. Even withthe use of a robotic system, since any succeeding liquid is added onto aliquid receiving zone 16 already wet with a preceding liquid, mixing ofthe two liquids invariably occurs, leading to unpredictable result,adversely affecting duration of any given step, preventing performanceof a truly sequential reaction, and affecting repeatability andaccuracy.

In U.S. Pat. No. 5,198,193 is taught a flow capillary device withmultiple capillary paths leading towards a single reaction zone, eachpath having a different length and/or a valve to allow variation oftiming of arrival of a liquid to the reaction zone. Such a device isineffective as at each intersection of capillary paths including twodifferent liquids, parallel flows are produced, analogous to theproduced when a succeeding liquid is added onto an already wet capillaryflow matrix as discussed above. Further, the valves described in such alateral flow capillary device are difficult to fabricate.

In European Patent No. EP 1044372 is taught a lateral flow capillarydevice where sample and reagent liquids are added at two or moreadjacent positions along a capillary flow matrix that is substantially astrip of bibulous material, e.g., 8 micron pore size polyester backednitrocellulose. N+1 narrow (e.g., 1 mm) spacers, impermeable hydrophobicstrips of material (mylar or polyester sticky tape) are placedperpendicularly to the flow direction to define N broad (e.g., 5 mm)liquid receiving zones upstream of a reaction zone located upstream of aliquid drain. When liquids are added simultaneously to the liquidreceiving zones a portion of each liquid is absorbed through the uppersurface of the capillary flow matrix at the liquid receiving zone.Liquid that is not immediately absorbed remains as drops on the surfaceof a respective liquid receiving zone, where adjacent drops areprevented from mixing or flowing along the surface of the capillary flowmatrix by the spacers. In cases where the liquids are addedsimultaneously an interface between the two liquids is formed in thevolume of the matrix underneath the spacer, while excess liquid remainson the surface of a liquid receiving zone. Liquid from a first, mostdownstream, liquid receiving zone is transported downstream by capillaryflow past the reaction zone to the liquid drain. When all the liquid inthe first liquid receiving zone is exhausted, the second liquidreceiving zone is transported downstream by capillary flow past thereaction zone to the liquid drain.

Seemingly the teachings of EP 1044372 provide the ability to performmultistep reactions using a lateral flow capillary device, butpractically the teachings are severely limited by limitations imposed bythe structure of the lateral flow capillary device.

A first limitation is that the amount of liquid added to a liquidreceiving zone is limited. The liquid is added as a drop resting on aliquid receiving zone. If the surface tension of the liquid isunsufficient, for example due to size or due to detergents in theliquid, if the capillary flow matrix is highly hydrophillic or if thelateral flow capillary device is perturbed, the drop collapses andspills from the lateral flow capillary device.

A second limitation is that the liquids must be added simultaneously. Ifliquids are added non-simultaneously, a liquid added to a first liquidreceiving zone flows into a second, adjacent, liquid receiving zone.When a second liquid is added to the second liquid receiving zone, thesecond liquid flows into a volume of the matrix from the top through dryparts of the second liquid receiving zone while the second liquid flowsinto the same volume laterally. The two liquids mix, and as discussedabove, leads to unpredictable result, adversely affects duration of agiven step, prevents performance of a truly sequential reaction, andaffects both repeatability and accuracy of the results.

A third limitation is that the teachings of EP 1044372 may lead to theformation of a multiple capillary paths. As noted above, a spacer is astrip of smooth material attached using adhesive to the top surface ofthe matrix that has micron scale features. As a result, capillary pathsare formed in the space between a spacer and the capillary flow matrixthrough which two liquids in adjacent liquid receiving zones may bemixed and as discussed above, leads to unpredictable result, adverselyaffects duration of a given step, prevents performance of a trulysequential reaction, and affects both repeatability and accuracy of theresults.

An additional disadvantage of the teachings of EP 1044372 is thereliance on adhesives for securing the spacers to the capillary flowmatrix. In the art it is known that adhesives, especiallynon-polymerizing adhesives, are attracted by and over time migrate intobibulous materials such as nitrocellulose that are suitable for use ascapillary flow matrices (see, for example, Kevin Jones; Anne Hopkins,Effect of adhesive migration in lateral flow assays; IVD Technology,September 2000). Thus, after a period of storage, the adhesive securinga spacer to a capillary flow matrix of a device made in accordance withthe teachings of EP 1044372 would migrate into the pores of thecapillary flow matrix in the region where the liquid-liquid interface isto form. The presence of a hydrophobic adhesive in the matrix blockspores or modify the capillary properties of the pores so that aninterface formed between liquids is indefinite and not clear, leading tomixing of the two liquids of the interface and concomitant negativeeffects. Another disadvantage of using adhesives is the possibledetachment of the spacers from the matrix during prolonged storage.

In U.S. Pat. No. 4,981,786 is taught a lateral flow capillary devicewith two reservoirs. The provision of a lateral flow capillary devicewith two or more reservoirs allows addition of two or more succeedingliquids without mutual contamination: once a liquid has been added to afirst reservoir, remnants of the liquid remain on the walls of thereservoir. Any liquid added through the same reservoir will becontaminated with the remnants. In a first lateral flow capillary devicetaught in U.S. Pat. No. 4,981,786, two or three distinct reservoirs arein fluid communication with a capillary flow matrix through distinct andphysically separated liquid receiving zones. Located at one of theliquid receiving zones is a reaction zone including a trapping reagent.A liquid drain is in capillary communication with capillary flow matrixdownstream from the two reservoirs. Although not entirely clear from thedescription, it is understood that the use of the first lateral flowcapillary device includes adding a small volume of sample through areservoir to provide a spot of sample at the reaction zone on thecapillary flow matrix and subsequently to add one or more reagents, eachreagent through a different reservoir.

In a second lateral flow capillary device taught in U.S. Pat. No.4,981,786, two distinct reservoirs are in fluid communication with acapillary flow matrix through distinct and physically separated liquidreceiving zones. In capillary communication with the upstream edge ofthe capillary flow matrix is a liquid reservoir that may be activated torelease a reagent liquid that subsequently migrates downstream. Areaction zone is located downstream from the two reservoirs. A liquiddrain is in capillary communication with capillary flow matrixdownstream from the reaction zone.

In both lateral flow capillary devices are taught a number of structuralfeatures to keep a capillary flow matrix in place but make only minimalcontact therewith. Further, it is noted that there is little or nocontact between a reservoirs and the capillary flow matrix at arespective liquid receiving zone, and if there is contact it is onlylight contact resulting from swelling of the capillary flow matrix uponwetting. Such features preclude the use of the lateral flow capillarydevices as effective devices for multistep reactions in a manneranalogous to the disclosed in EP 1044372. When a first liquid is addedto a first reservoir and simultaneously a second liquid is added to asecond adjacent upstream reservoir, the first and second liquids bothflow into the capillary flow matrix through a respective liquidreceiving zone. When the two liquids meet, an interface is formed andthe first liquid begins to flow downstream. Uncontrollably, liquidbegins to leak from the capillary flow matrix at any point where analternate capillary path exists, for example down the supportingstructures on which the capillary flow matrix rests or along thelaterally disposed walls that hold the capillary flow matrix in place.Liquid also climbs up any object contacting the upper surface of thecapillary flow matrix, for example where a reservoir contacts thecapillary flow matrix. As a result, liquid leaks away from all liquidreceiving zones through any alternative capillary path, filling thelateral flow capillary device with liquid and rendering results of anexperiment useless.

It would be highly advantageous to have a lateral flow capillary deviceor methods for using lateral flow capillary devices for the performanceof multistep reactions in the fields of biology and medicine,particularly for diagnosis not having at least some of the disadvantagesof the prior art.

SUMMARY OF THE INVENTION

Embodiments of the present invention successfully address at least someof the shortcomings of the prior art by providing a lateral flowcapillary device and a method including the use of a lateral flowcapillary device allowing performance of multistep reactions.Embodiments of the present invention allow performance of multistepreactions such as multistep binding assays accurately and repeatablyeven in non-laboratory conditions and even by less skilled operators.

According to the teachings of the present invention there is provided alateral flow capillary device comprising: a) a unipath bibulouscapillary flow matrix having an upstream end and a downstream enddefining a flow direction; b) at least two reservoirs in fluidcommunication with the capillary flow matrix each through at least onerespective liquid receiving zone; wherein a reservoir contacts arespective liquid receiving zone through an opening constituting ahollow conduit having a rim pressing the matrix and wherein a portion ofthe capillary flow matrix between the two rims is an interface creationzone.

In embodiments of the present invention, the pressing is such thatliquid-induced swelling of the matrix is constrained, that is when thematrix is wet and swells, the rims apply pressure resisting theswelling.

In embodiments of the present invention, the rims press the matrix whenthe matrix is dry. In embodiments of the present invention the rims arepressed into the matrix when the matrix is dry.

In embodiments of the present invention, the rims are substantiallyparallel to the flow direction.

In embodiments of the present invention, pressure applied by a rim issubstantially uniform about the entire surface of the rim.

In embodiments of the present invention, the matrix is substantiallycompressible, that is does not break under pressure which leads to areduction in volume of the matrix yet substantially retains structuralintegrity. In embodiments of the present invention, the internalsurface-area volume⁻¹ of the matrix proximate to a rim is higher thandistant from the rim.

In embodiments of the present invention, the matrix comprises or evenessentially consists of glass fibers and/or nitrocellulose and/or porouspolyethylene.

In embodiments of the present invention, opposite each rim is disposed asupporting component supporting the matrix against the pressing.

In embodiments of the present invention, the matrix is suspended betweenthe rims and the supporting components.

In embodiments of the present invention the matrix is attached to asubstantially impermeable backing. In embodiments of the presentinvention, the impermeable backing contacts at least one supportingcomponent supporting the impermeable backing against the pressing. Inembodiments of the present invention, opposite each rim is disposed asupporting component supporting the matrix against the pressing. Inembodiments of the present invention, the matrix is suspended betweenthe rims and the supporting components.

In embodiments of the present invention, the lateral flow capillarydevice further comprises downstream from at least one liquid receivingzone, a reaction zone comprising at least one capturing entity (e.g., amember of a specific binding pair) configured to capture a material(e.g., an analyte or a product of a reaction involving the analyte)flowing through the capillary flow matrix. In embodiments, the reactionzone is in a liquid receiving zone of a reservoir.

In embodiments of the present invention, the lateral flow capillarydevice further comprises downstream from at least two liquid receivingzones, a reaction zone comprising at least one capturing entityconfigured to capture a material flowing through the capillary flowmatrix.

In embodiments of the present invention, the lateral flow capillarydevice further comprises a liquid drain in fluid communication with thecapillary flow matrix downstream from at least two of the at least tworeservoirs.

In embodiments of the present invention, the fluid communication throughthe liquid receiving zones is non-capillary communication.

In embodiments of the present invention, the interface creation zone isa volume of matrix with a length in the flow direction, and of a widthand height substantially of the capillary flow matrix, that is theinterface creation zone corresponds to a cross section of the matrixwith a finite length. In embodiments of the present invention, theinterface creation zone has a length of at least about 50%, at leastabout 75%, at least about 100%, even at least about 150%, and even atleast about 400% of a dimension of a liquid receiving zone in the flowdirection.

In embodiments of the device of the present invention, liquid inducedswelling of the interface creation zone is unconstrained.

In embodiments of the present invention, a reservoir is substantially acontainer.

In embodiments of the present invention, the device further comprises ahousing containing the capillary flow matrix. In embodiments of thepresent invention, sides of the capillary flow matrix are substantiallydevoid of contact with the housing.

According to the teachings of the present invention there is alsoprovided a device useful for preparation of lateral flow capillarydevice, comprising: a) a first component, including a reservoir with atleast one wall configured to hold liquids and a lowest area, the lowestarea defined by a non-capillary opening defining a hollow conduit with arim and at least one extension protruding from an outer surface of thewall; and b) a second component, including a body with a counter-supportplatform at a top-end and at least one extension protruding from thebody wherein an extension of the first component and a extension of thesecond component are configured to mutually engage so that the rim andthe counter-support platform are spaced apart and substantiallyparallel.

In embodiments of the present invention, the opening is a non-capillaryopening, that is of dimensions that are not conducive to capillary flowtherethrough.

In embodiments of the present invention, the opening has across-sectional area of at least about 1 mm², of at least about 3 mm² oreven a cross-sectional area of at least about 7 mm².

In embodiments of the present invention, the first component and thesecond component each comprise at least two extensions.

In embodiments of the present invention, at least one first componentextension and at least one second component extension together define ahinge when engaged.

In embodiments of the present invention, the mutual engaging includesinterlocking, for example, by snapping together.

According to the teachings, of the present invention there is alsoprovided a kit for assembly of a lateral flow capillary device,comprising: a) a unipath bibulous capillary flow matrix having athickness; and b) at least two devices as described above, wherein thedistance is sufficient so that a rim contacts the matrix when the twocomponents are engaged about the matrix. In embodiments of the presentinvention, the distance is sufficient to clamp the matrix so as to pressthe rim into the matrix perpendicularly to the thickness when the twocomponents are engaged. In embodiments of the present invention, thematrix is substantially a strip of material, for example comprisingglass fiber.

In embodiments of the present invention, the matrix is attached to asubstantially impermeable backing. In embodiments of the presentinvention, the backing is substantially planar. In embodiments of thepresent invention, the matrix together with the backing aresubstantially a strip.

In embodiments of the present invention, the capillary flow matrixincludes a reaction zone comprising at least one capturing entity (e.g.,a member of a specific binding pair) configured to capture a material(e.g., an analyte or a product of a reaction involving the analyte)flowing through the capillary flow matrix.

According to the teachings of the present invention there is alsoprovided a method of performing a reaction comprising: a) providing alateral flow capillary device as described above; b) adding a firstamount of a first liquid to a first reservoir so that the first liquidflows into the capillary flow matrix through the respective liquidreceiving zone; and c) adding a second amount of a second liquid to asecond the reservoir so that a second liquid flows into the capillaryflow matrix through a respective liquid receiving zone; so that a staticliquid-liquid interface is formed between the first liquid and a liquidin an interface creation zone; wherein the first amount and the secondamount are such that first liquid substantially remains in the firstreservoir and second liquid substantially remains in the secondreservoir subsequent to formation of the static interface; and whereinthe interface begins to move only subsequent to exhaustion of a liquidfrom a reservoir. Generally, the interface moves downstream uponexhaustion of the more downstream reservoir.

Generally, the static liquid-liquid interface is formed between thefirst liquid and the second liquid in the interface creation zonebetween the respective reservoirs to which the two liquids were added.

In embodiments, the static liquid-liquid interface is formed between thefirst liquid and a liquid in the matrix that is located between theliquid receiving zone associated with the first reservoir and the liquidreceiving zone associated with the second reservoir. Such a situationoccurs, for example when using a device provided with three reservoirsand three respective liquid receiving zones where in the reservoirassociated with the most downstream liquid receiving zone is added anamount of the first liquid and in the reservoir associated with the mostupstream liquid receiving zone is added an amount of the second liquidso that in neither case does all the liquid enter the matrix, but in thereservoir of the middle receiving zone is added an amount of a thirdliquid that entirely enters the matrix. In such a case two interfacesare formed: one between the first liquid and the third liquid and onebetween the third liquid and the second liquid.

In embodiments of the present invention, the first liquid and the secondliquid are substantially identical, e.g., both are analyte containingsample. In embodiments of the present invention, the first liquid andthe second liquid are substantially different, for example one is ananalyte containing sample and one is a reagent liquid (e.g., a solutionincluding signal producing label).

In embodiments of the present invention, the first amount and the secondamount are substantially different. In embodiments of the presentinvention, the first amount and the second amount are substantiallyequal.

In embodiments of the present invention, adding of the second amount issubsequent to the adding of the first amount. In embodiments of thepresent invention, adding of the first amount and of the second amountis substantially simultaneous.

According to the teachings of the present invention there is alsoprovided a method of performing a reaction comprising: a) providing alateral flow capillary device as described above including: i) on thecapillary flow matrix, a first liquid receiving zone in fluidcommunication with a first reservoir; ii) on the capillary flow matrixupstream of the first liquid receiving zone, a second liquid receivingzone in fluid communication with a second reservoir; iii) a firstreagent disposed at a location inside the first reservoir and/or in thecapillary flow matrix in proximity to the first liquid receiving zone ordownstream therefrom; iv) a second reagent disposed at a location insidethe second reservoir and/or in the capillary flow matrix in proximity tothe second liquid receiving zone; b) adding a first amount of a firstliquid to the first reservoir so that the first liquid flows into thecapillary flow matrix through the first liquid receiving zone to contactthe first reagent; c) adding a second amount of a second liquid to thesecond reservoir so that the second liquid flows into the capillary flowmatrix through the second liquid receiving zone to contact the secondreagent; so that a static interface is formed between the first liquidand the second liquid in the interface creation zone; wherein the firstamount and the second amount are such that liquid substantially remainsin the first reservoir and in the second reservoir subsequent toformation of the static interface; and wherein the interface begins tomove only subsequent to exhaustion of the liquid from a reservoir.

In embodiments of the present invention, the first liquid and the secondliquid are substantially identical, e.g., both are analyte containingsample. In embodiments of the present invention, the first liquid andthe second liquid are substantially different, for example one is ananalyte containing sample and one is a reagent liquid (e.g., a solutionincluding signal producing label).

In embodiments of the present invention, the first amount and the secondamount are substantially different. In embodiments of the presentinvention, the first amount and the second amount are substantiallyequal.

In embodiments of the present invention, downstream of the first liquidreceiving zone on the capillary flow matrix is a reaction zonecomprising at least one capturing entity configured to capture amaterial flowing through the capillary flow matrix.

In embodiments of the present invention, adding of the second amount andthe adding of the first amount is sequential. In embodiments of thepresent invention, adding of the first amount and of the second amountis substantially simultaneous.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention.

In the drawings:

FIG. 1 (prior art) depicts a one reservoir lateral flow capillarydevice;

FIG. 2 schematically depicts an embodiment of a lateral flow capillarydevice of the present invention including three liquid reservoirs and acapillary flow matrix without a backing, in cross section;

FIG. 3 schematically depicts the formation of standing columns of liquidin a lateral flow capillary device in accordance with the method of thepresent invention;

FIG. 4 schematically depicts an embodiment of a lateral flow capillarydevice of the present invention including four liquid reservoirs and acapillary flow matrix with a backing, in cross section;

FIG. 5A schematically depicts an embodiment of a device of the presentinvention useful for preparing a lateral flow capillary device;

FIG. 5B schematically depicts a top view of a lateral flow capillarydevice made of an assembled kit of the present invention including twodevices of FIG. 5A and a capillary flow matrix of plastic backed glassfiber;

FIG. 5C schematically depicts a side view of a lateral flow capillarydevice made of an assembled kit of the present invention including twodevices of FIG. 5A and capillary flow matrix of plastic backed glassfiber

FIG. 6A schematically depicts an embodiment of a lateral flow capillarydevice of the present invention, exploded in cross section to showcomponents;

FIG. 6B schematically depicts of a capillary flow matrix of the lateralflow capillary device of FIG. 6A;

FIG. 6C is a schematic depiction of the lateral flow capillary device ofFIG. 6A, assembled in cross section;

FIG. 7A is the result of experiment 2, described below, comparingdetection of an analyte in accordance with the teachings of the presentinvention that depicts the result obtained with the first lateral flowcapillary device;

FIG. 7B is the result of experiment 2, described below, comparingdetection of an analyte in accordance with the teachings of the presentinvention that depicts the result obtained with the second lateral flowcapillary device;

FIG. 7C is the result of experiment 2, described below, comparingdetection of an analyte in accordance with the teachings of the presentinvention that depicts the result obtained with the third lateral flowcapillary device;

FIG. 8A and FIG. 8B are results of experiment 3 described below,comparing detection of an analyte in accordance with the teachings ofthe present invention; FIG. 8C and FIG. 8D are results of experiment 3using a single reservoir lateral flow capillary device;

FIG. 9 are results of experiment 4 described below, a calibration curvefor 11-dehydro-TxB2−competition assay acquired in accordance with theteachings of the present invention; and

FIG. 10 are results of experiment 10 described below, showing thecorrelation between the intensity of fluoresence emitted by a reactionzone of a lateral flow capillary device of the present invention and thetotal volume of liquid added.

PREFERRED EMBODIMENTS OF THE INVENTION

The present invention is of a lateral flow capillary device and methodsof using the lateral flow capillary device. The present invention allowsperformance of effective and repeatable multistep reactions such asmultistep specific binding assays for example for serological testing.

In the description the embodiments are directed to an analytical methodfor detecting an analyte that is a biomarker such as an antigen,antibody, metabolite, toxicant or other detectable material from humanor other living source such as blood, urine, tissue, or from anon-living source such as an environmental source like water, soil orsewage. In the description the embodiments are directed to binding theanalyte to an anti-analyte immobilized at a reaction zone on thecapillary flow matrix which together with the analyte constitutes aspecific binding pair such as an antibody, antigen, DNA or otherspecific binding pair (sbp) member and that the bound analyte is thendetected directly or by a labeled reagent producing a detectable signalor that produces a detectable signal after being exposed to a thirdreagent which reacts with the labeled reagent and produces a detectablesignal that can be visualized or measured, by reading instrument.

The principles, uses and implementations of the teachings of the presentinvention may be better understood with reference to the accompanyingdescription, figures and examples, perusal of which allows one skilledin the art to implement the teachings of the present invention withoutundue effort or experimentation. In the figures, like reference numeralsrefer to like parts throughout.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth herein. The invention can be implemented withother embodiments and can be practiced or carried out in various ways.It is also understood that the phraseology and terminology employedherein is for descriptive purpose and should not be regarded aslimiting.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include techniques from the fields ofbiology, diagnostics engineering, material science and physics. Suchtechniques are thoroughly explained in the literature.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. In addition, the descriptions,materials, methods and examples are illustrative only and not intendedto be limiting. Methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention.

As used herein, the terms “comprising” and “including” or grammaticalvariants thereof are to be taken as specifying the stated features,integers, steps or components but do not preclude the addition of one ormore additional features, integers, steps, components or groups thereof.This term encompasses the terms “consisting of” and “consistingessentially of”.

The phrase “consisting essentially of” or grammatical variants thereofwhen used herein are to be taken as specifying the stated features,integers, steps or components but do not preclude the addition of one ormore additional features, integers, steps, components or groups thereofbut only if the additional features, integers, steps, components orgroups thereof do not materially alter the basic and novelcharacteristics of the claimed composition, device or method.

The term “method” refers to manners, means, techniques and proceduresfor accomplishing a given task including, but not limited to, thosemanners, means, techniques and procedures either known to, or readilydeveloped from known manners, means, techniques and procedures bypractitioners of the relevant arts. Implementation of the methods of thepresent invention involves performing or completing selected tasks orsteps manually, automatically, or a combination thereof.

Herein, the term “analyte” refers to the compound or composition to bedetected or quantitatively analyzed and which has at least one epitopeor binding site. An analyte can be any substance for which there exist anaturally occurring analyte specific binding member or for which ananalyte-specific binding member can be prepared. e.g., carbohydrate andlectin, hormone and receptor, complementary nucleic acids, and the like.Further, possible analytes include virtually any compound, composition,aggregation, or other substance which may be immunological detected.That is, the analyte, or portion thereof, will be antigenic or haptenichaving at least one determinant site, or will be a member of a naturallyoccurring binding pair.

Analytes include, but are not limited to, toxins, organic compounds,proteins, peptides, microorganisms, bacteria, viruses, amino acids,nucleic acids, carbohydrates, hormones, steroids, vitamins, drugs(including those administered for therapeutic purposes as well as thoseadministered for illicit purposes), pollutants, pesticides, andmetabolites of or antibodies to any of the above substances.

Generally an analyte is found in a “sample” and the teachings of thepresent invention are applied to the sample to determine the presence ofor an amount of analyte present in a sample.

Herein the term “sample” refers to anything which may contain an analytefor which an analyte assay is desired. The sample may be a biologicalsample, such as a biological fluid or a biological tissue. Examples ofbiological fluids include urine, blood, plasma, serum, saliva, semen,stool, sputum, cerebral spinal fluid, tears, mucus, amniotic fluid orthe like. Biological tissues are aggregate of cells, usually of aparticular kind together with their intercellular substance that formone of the structural materials of a human, animal, plant, bacterial,fungal or viral structure, including connective, epithelium, muscle andnerve tissues. Examples of biological tissues also include organs,tumors, lymph nodes, arteries and individual cell(s). In addition, asolid material suspected of containing the analyte can be used as thetest sample once it is modified to form a liquid medium or to releasethe—analyte. Pretreatment may involve preparing plasma from blood,diluting viscous fluids, and the like. Methods of treatment can involvefiltration, distillation, separation, concentration, inactivation ofinterfering components, and the addition of reagents. Besidesphysiological fluids, other samples can be used such as water, foodproducts, soil extracts, and the like for the performance of industrial,environmental, or food production assays as well as diagnostic assays.The selection and pretreatment of biological, industrial, andenvironmental samples prior to testing is well known in the art and neednot be described further.

As used herein, the term “specifically binds” refers to the bindingspecificity of a “Specific binding pair member” which is a member of aspecific binding pair, i.e., two different molecules wherein one of themolecules specifically binds with the second molecule through chemicalor physical means. The two molecules are related in the sense that theirbinding with each other is such that they are capable of distinguishingtheir binding partner from other assay constituents having similarcharacteristics. The members of the specific binding pair are referredto as ligand and receptor (anti ligand), sbp member and sbp partner, andthe like. A molecule may also be a sbp member for an aggregation ofmolecules; for example an antibody raised against an immune complex of asecond antibody and its corresponding antigen may be considered to be ansbp member for the immune complex.

In addition to antigen and antibody specific binding pair members, otherspecific binding pairs include, as examples without limitation, biotinand avidin, carbohydrates and lectins, complementary nucleotidesequences, complementary peptide sequences, effector and receptormolecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes,a peptide sequence and an antibody specific for the sequence or theentire protein, polymeric acids and bases, dyes and protein binders,peptides and specific protein binders (e.g., ribonuclease, S-peptide andribonuclease S-protein), metals and their chelators, and the like.Furthermore, specific binding pairs can include members that are analogsof the original specific binding member, for example an analyte-analogor a specific binding member made by recombinant techniques or molecularengineering.

A sbp member is analogous to another sbp member if they are both capableof binding to another identical complementary sbp member. Such a sbpmember may, for example, be either a ligand or a receptor that has beenmodified by the replacement of at least one hydrogen atom by a group toprovide, for example, a labeled ligand or labeled receptor. The sbpmembers can be analogous to or complementary to the analyte or to an sbpmember that is complementary to the analyte. If the specific bindingmember is an immunoreactant it can be, for example, an antibody,antigen, hapten, or complex thereof. If an antibody is used, it can be amonoclonal or polyclonal antibody, a recombinant protein or antibody, achimeric antibody, a mixture(s) or fragment (s) thereof, as well as amixture of an antibody and other specific binding members. The detailsof the preparation of such antibodies and their suitability for use asspecific binding members are known to those skilled in the art.

“Labeled reagent” refers to a substance comprising a detectable labelattached with a specific binding member. The attachment may be covalentor non-covalent binding, but the method of attachment is not critical tothe present invention. The label allows the label reagent to produce adetectable signal that is related to the presence of analyte in thesample. The specific binding member component of the label reagent isselected to directly bind to the analyte or to indirectly bind theanalyte by means of an ancillary specific binding member, which isdescribed in greater detail hereinafter.

In addition, the specific binding member may be labeled before orduring, the performance of the assay by means of a suitable attachmentmethod.

“Label” refers to any substance which is capable of producing a signalthat is detectable by visual or instrumental means. Various labelssuitable for use in the present invention include labels which producesignals through either chemical or physical means. Such labels caninclude enzymes, fluorescent compounds, chemiluminescent compounds, andradioactive labels. Other suitable labels include particulate labelssuch as colloidal metallic particles such as gold, colloidalnon-metallic particles such as selenium, dyed or colored particles suchas a dyed plastic or a stained microorganism, organic polymer latexparticles and liposoms, colored beads, polymer microcapsules, sacs,erythrocytes, erythrocyte ghosts, or other vesicles containing directlyvisible substances, and the like. Typically, a visually detectable labelis used as the label component of the label reagent, thereby providingfor the direct visual or instrumental readout of the presence or amountof the analyte in the test sample without the need for additional signalproducing, components at the detection sites.

The selection of a particular label is not critical to the presentinvention, but the label will be capable of generating a detectablesignal either by itself, or be instrumentally detectable, or bedetectable in conjunction with one or more additional signal producingcomponents.

A variety of different label reagents can be formed by varying eitherthe label or the specific binding member component of the label reagent;it will be appreciated by one skilled in the art that the choiceinvolves consideration of the analyte to be detected and the desiredmeans of detection. As discussed below, a label may also be incorporatedused in a control system for the assay.

For example, one or more signal producing components can be reacted withthe label to generate a detectable signal. If the label is an enzyme,then amplification of the detectable signal is obtained by reacting theenzyme with one or more substrates or additional enzymes and substratesto produce a detectable reaction product.

Labeled enzymes used in the field include, for example, Alkalinephosphatase, Horseradish peroxidase, Glucose oxidase and Urease.

In an alternative signal producing system, the label can be afluorescent compound where no enzymatic manipulation of the label isrequired to produce the detectable signal. Fluorescent moleculesinclude, for example, fluorescein, phycobiliprotein, rhodamine and theirderivatives and analogs are suitable for use as labels in such a system.

The use of dyes for staining biological materials, such as proteins,carbohydrates, nucleic acids, and whole organisms is documented in theliterature. It is known that certain dyes stain particular materialspreferentially based on compatible chemistries of dye and ligand. Forexample, Coomassie Blue and Methylene Blue for proteins, periodicacid-Schiffs reagent for carbohydrates, Crystal Violet, SafraninO, andTrypan Blue for whole cell stains, Ethidium bromide and Acridine Orange

“Signal producing component” refers to any substance capable of reactingdirectly or indirectly with the labeled reagent to produce signal thatis detectable by visual or instrumental mean. The component may besubstrate catalyzed by the labeled enzyme or dyes that may reactchemically with the label reagent (dsDNA/Acridine Orange), enzymessubstrate such as: BCIP/NBT; Azonaphtol phosphate; 3-AEC;4-chloronaphyhol; tetrazolium salt/PMS; Urea/PH indicators.

In embodiments of the present invention, a capillary flow matrixincludes at least one reaction zone. A reaction zone is a region orvolume of the matrix comprising at least one capturing entity configuredto capture a material flowing through the capillary flow matrix indefined regions for conducting the assay reaction including a test lineand a control line. A “test line” Is the region in the reaction zone, inwhich the analytical assay is performed. The region comprises specificbinding pair (sbp) member which is immobilized to the matrix of thecapillary path. The sbp member can be an antibody or antigen nucleicacid or modifications of the above. It may by proteins like avidin andits derivatives or saccharides such has lectins. Which are part ofbinding pair being capable of binding directly or indirectly the analyteof interest. Several test line may be in the reaction zone each of adistinct specific binding pair for different analytes. A “control line”Is the region in the reaction zone, in which a reaction for confirmingthe validity of the assay is performed; the control line may also be acalibration line or lines for correction of the assay signals (results)obtained in the test line. The control line comprise of immobilized spbmember with binding abilities to one or more of the reagentsparticipating in the reaction or compounds existing in the sample.

In the present invention, a capillary flow matrix is bibulous, that iscomprises a bibulous, porous or other cavity shaped material allowingcapillary transport of liquids therethrough, that is the pores define acontinuous system of capillary flow channels. Generally, for aqueousliquids capillary transport requires a continuous path of pores of lessthan about 2 mm in size, generally in the range of 0.05 microns to 100microns. As is described herein, a suitable capillary flow matrix issubstantially compressible, that is to say, retains structural integrityand does not break under applied pressure which leads to a reduction involume, for example pressure applied by the reservoir rims. Inembodiments, pressure applied to a capillary flow matrix perpendicularlyby a reservoir rim compresses the capillary flow matrix to substantiallysame extent through the entire height of the capillary flow matrix. Inembodiments, a matrix is thick enough and soft enough so thatcompression caused by applied pressure is local to the pressing. Inembodiments, pressure applied by a rim to a capillary flow matrixsubstantially compresses the matrix and reduces the internalsurface-area volume⁻¹ to a depth of no more than 40% of the thickness.

“Bibulous material” include but are not limited to materials composed ofglass fiber paper or derivatized glass fiber paper, cellulose and itsderivatives, nylons, PVDF, polysulfones, PTFE and polypropylene, paperand derivatized paper, see Eric Jallerat and Volkmar Thom, “Filtermembranes and bioseparation equipment and supplies” by IVD Technology(2004) or catalogues of manufacturers such as Millipor Corp. (Bedford,Mass., USA), Watman Inc. (New Jersey, USA) or Ahlstrom Corp. (Helsinki,Finland).

Typically, the bibulous member consists of a series of fibers drawntogether in parallel to form an open wick with some mechanical integritydue to bonding between the fibers, with the space between the fibersacting to form channels, which draw up liquid. Suitable fibers includepolyester, polyamides such as nylons, and bicomponent fibers such aspolyethylene/polyester, nylon/polyester and the like. Bicomponentpolyethylene/polyester fibers typically comprise a polyester centralcore with an external sheath of polyethylene. Inherently hydrophobicfibers such as polypropylenes can also be used provided they are waterwettable or, if necessary, are rendered water wettable by othercomponents such as surfactants or hydrophilic polymers. In principle anywettable fiber is suitable.

Fibers can be formed into a bibulous member by a variety of processes,such as annealing to partially melt the surface/sheath region and causeinterpenetration of the polymer chains, which set on cooling see.Alternatively, adhesives, such as latex adhesives, may be used.

Capillary matrices of embodiments of the invention are of various formsincluding but not limited to sheets, columns, membranes, and compressedfibers. Suitable materials include but are not limited to porousmaterials and fibrous materials, including woven, rationally orientedand randomly oriented fibrous materials. Suitable materials includepolymeric materials such as porous polymers including porouspolyethylene, polypropylene, polytetrafluoroethylene (PTFE), ethylenevinyl acetate (EVA), polyether sulfone (PS), thermoplastic urethane(TPU), polyamide (e.g., Nylon 6) and copolymers thereof such as porouspolymers manufactured by the Porex Corporation, Fairburn Ga., USA.Suitable materials include fibrous materials such as cellulose,cellulosic materials, cellulose derivatives, glass fibers, paper, filterpaper, chromatographic paper, synthetic or modified naturally occurringpolymers, such as nitrocellulose, cellulose acetate and cotton.

In embodiments, a bibulous capillary flow matrix of the presentinvention is a uniform structure such as strip of paper or a combinationof several materials comprising a unipath structure. In embodiments acapillary flow matrix of the present invention is attached to asubstantially impermeable backing material, for example as is known inthe field of thin-layer chromatography where porous fibrous matter isbound to a solid impermeable backing. For example, generally a backingis of the same dimensions as the capillary flow matrix: when smaller orlarger then the interface between the matrix and backing may produce acapillary path parallel to the capillary path defined by the capillaryflow matrix. Suitable materials from which to form a backing include butare not limited to polyethylene, polypropylene, poly(4-methylbutene),polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon,poly(vinyl butyrate), glass, ceramics, metals, polyurethane, neoprene,latex and silicone rubber.

A material exceptionally suitable for preparing a capillary flow matrixof the present invention is glass fiber especially plastic backed glassfiber, including glass fiber derivative such as glassfiber/cellulose/polyester matrices. Glass fiber membranes are relativelythick, (typically up to 2 mm), have pore sizes of 1-40 micron and arelatively high water flow rate (when compared to typical nitrocellulosematrix) allowing large sample and reagent flow through. An additionaladvantage of glass fiber, as noted above, is that glass fiber isrelatively thick, soft and compressible so that when pressure is appliedin accordance with the teachings of the present invention, compressionis local to the point of pressure.

A material exceptionally suitable for preparing a capillary flow matrixof the present invention is nitrocellulose, especially plastic backednitrocellulose, especially having a pore size of between 0.45 and 15micron.

A material exceptionally suitable for preparing a capillary flow matrixof the present invention is porous polyethylene, especially having apore size of between 0.2 and 20 micron, preferably between 1 and 12micron, available from the Porex Corporation, Fairburn Ga., USA.

The actual physical size of a capillary flow matrix of a lateral flowcapillary device of the present invention is determined by many factorsespecially the material from which the matrix is made and the specificuse or uses for which the lateral flow capillary device is intended.That said, in some embodiments a lateral flow capillary device of thepresent invention is a manually operated lateral flow capillary device.In embodiments of the present invention, it is generally preferred thatthe length of a capillary flow matrix be convenient for manual use andstorage of a lateral flow capillary device, which is to say generallybut not necessarily at least about 1 cm, and generally not greater thanabout 30 cm, not greater than about 15 cm and even not greater thanabout 10 cm.

In embodiments of the present invention, it is generally preferred thatthe width of a capillary flow matrix be sufficiently narrow to allowconcentration of a material producing a signal in a small area toincrease the contrast of a signal produced and reduce the liquidcapacity of the capillary flow matrix, but also sufficiently wide toallow simple observation of the signal by a user, which is to saygenerally but not necessarily between about 1 mm and 20 mm.

In embodiments of the present invention, it is generally preferred thatthe capillary flow matrix be relatively thick to allow a high liquidcapacity and flow rate and also ensure that the pressure applied by therims compresses the matrix only locally. Preferably the capillary flowmatrix is up to about 2 mm thick, up to about 1 mm thick, and preferablybetween about 0.05 and about 0.5 mm thick.

In embodiments of the present invention a capillary flow matrix is incapillary communication with a liquid drain. A liquid drain is generallya component made of a bibulous material and having a liquid absorbingcapacity that is significantly larger than that of a respectivecapillary flow matrix. In embodiments of the present invention, a liquiddrain is integrally formed with a respective capillary flow matrix. Inembodiments of the present invention, a liquid drain constitutes atleast one component distinct from a respective capillary flow matrix. Inembodiments of the present invention, a liquid drain is of a shape or ofmaterial that allows a faster rate of capillary movement than through arespective capillary flow matrix. Suitable materials from which tofashion a drain are described, for example, in U.S. Pat. No. 4,632,901,such as, for example, fibrous materials such as cellulose acetatefibers, cellulose or cellulose derivatives, polyester, or polyolefins.

In embodiments of the present invention, reservoirs are of any suitableshape or size. As a joint between two faces may define a capillarychannel, in embodiments, the inner surface of a reservoir in contactwith or immediate proximity with a liquid receiving zone is continuous,for example circular, oval or otherwise curved. The volume of a givenreservoir is determined by many factors and the exact implementation ofa respective lateral flow capillary device. That said, a typicalreservoir of a lateral flow capillary device of the present inventiongenerally has a capacity of at least about 5 microliter, at least about20 microliter or even at least about 50 microliter and generally nogreater than about 5000 microliter, no greater than about 1000microliter and even no greater than about 300 microliter.

Lateral Flow Capillary Device of the Present Invention

An embodiment of a lateral flow capillary device of the presentinvention, 24 is depicted in FIG. 2. Lateral flow capillary device 24includes a unipath bibulous capillary flow matrix 18 having an upstreamend 26 and a downstream end 28 defining a flow direction 30. Capillaryflow matrix 18 of lateral flow capillary device 24 is substantiallyporous membrane of enforced nitrocellulose devoid of a backing layer.

Three reservoirs, a downstream reservoir 32 a, a middle reservoir 32 band an upstream reservoir 32 c constituting open-topped containers, arein non-capillary fluid communication (to preserve the unipath of lateralflow capillary device 24) with capillary flow matrix 18 each through arespective liquid receiving zone 34 a, 34 b and 34 c. The distancebetween the edges of liquid receiving zones 34 a, 34 b and 34 c is atleast 50% of the dimension of such a liquid receiving zone in flowdirection 30, although in embodiments is substantially larger. Thecapillary matrix between any two receiving zones is defined as aninterface creation zone. Interface creation zone 35 a is found betweenliquid receiving zones 34 a and 34 b. Interface creation zone 35 b isfound between liquid receiving zones 34 b and 34 c.

Each reservoir 32 a, 32 b or 32 c contacts a respective liquid receivingzone 34 a, 34 b and 34 c through an opening at the bottom of thereservoir constituting a hollow conduit having a rim 36 a, 36 b and 36c. Opposite each rim 36 a, 36 b and 36 c are disposed supportingcomponents 38 a, 38 b and 38 c respectively Rims 36 a, 36 b and 36 cpress into capillary flow matrix 18, even when capillary flow matrix isdry while supporting components 38 a, 38 b and 38 c support capillaryflow matrix 18 against the pressing so that capillary flow matrix 18 issubstantially suspended between rims 36 a, 36 b and 36 c and supportingcomponents 38 a, 38 b and 38 c Rims 36 a, 36 b and 36 c and the uppersurfaces of supporting components 38 a, 38 b and 38 c are substantiallyparallel to flow direction 30, ensuring that pressure applied by a rim36 a, 36 b or 36 c is substantially uniform about the entire surface ofthat rim.

As capillary flow matrix 18 is substantially compressible, the internalsurface-area per unit volume of matrix 18 proximate to a rim 36 a, 36 bor 36 c is higher than distant from a rim 36 a, 36 b or 36 c. Thepresumed significance of this difference is discussed hereinbelow. Thematrix material in interface creation zones 35 a and 35 b isunconstrained for liquid-induced swelling.

Capillary flow matrix 18 is provided with a reaction zone 20 includingat least one capturing entity configured to capture a material such asan analyte or a product of a reaction involving the analyte flowingthrough capillary flow matrix 18 downstream from liquid receiving zones34 a, 34 b and 34 c.

In proximity of downstream end 28, capillary flow matrix 18 is in fluidcommunication with liquid drain 23.

Capillary flow matrix 18 and liquid drain 23 are substantially containedwithin a housing 40 such that all sides of capillary flow matrix 18 aresubstantially devoid of contact with housing 40. Through housing 40above reaction zone 20 is an observation window 22 which in embodimentsis simply a gap through housing 40.

For use, in accordance with the method of the present invention, a firstamount of a first liquid, for example sample containing analyte isplaced in downstream reservoir 32 a, flows into capillary flow matrix 18through liquid receiving zone 34 a and spreads both upstream anddownstream from liquid receiving zone 34 a. A second amount of a secondliquid, for example labeled reagent, is placed in middle reservoir 32 b,flows into capillary flow matrix 18 through liquid receiving zone 34 band spreads both upstream and downstream from liquid receiving zone 34b. A third amount of a third liquid, for example signal producingcomponent, is placed in upstream reservoir 32 c, flows into capillaryflow matrix 18 through liquid receiving zone 34 c and spreads bothupstream and downstream from liquid receiving zone 34 c.

The third liquid flows upstream until all of capillary matrix 18upstream of liquid receiving zone 34 c becomes saturated with thirdliquid. Third liquid flows downstream from liquid receiving zone 34 cuntil encountering second liquid flowing upstream from liquid receivingzone 34 b forming a static interface 42 b (see FIG. 3) somewhere ininterface creation zone 35 b wherethrough there is substantially nomixing of liquids. Once interface 42 b is formed, and assuming that theamount of second and third liquids added is sufficient so as not to beentirely absorbed into capillary flow matrix 18, a standing column ofthird liquid is formed in upstream reservoir 32 c as third liquid isprevented from flowing downstream by pressure applied by second liquidin middle reservoir 32 b.

Second liquid flows downstream from liquid receiving zone 34 b untilencountering first liquid flowing upstream from liquid receiving zone 34a forming a static interface 42 a somewhere in interface creation zone35 a wherethrough there is substantially no mixing of liquids. Onceinterface 42 a is formed, and assuming that the amount of first andsecond liquids added is sufficient so as not to be entirely absorbedinto capillary flow matrix 18, a standing column of second liquid isformed in middle reservoir 32 b, the second liquid prevented fromflowing downstream by pressure applied by first liquid in downstreamreservoir 32 a.

First liquid from downstream reservoir 32 a drains down through liquidreceiving zone 34 a and flows downstream in flow direction 30 pastreaction zone 20 on which sbp member (receptor) is immobilized. Analyteif present in the sample is captured by the sbp member while liquid isabsorbed into liquid drain 23.

When all first liquid is drained from downstream reservoir 32 a, thefirst liquid/second liquid interface begins to move downstream in flowdirection 30 as second liquid from middle reservoir 32 b drains downthrough liquid receiving zone 34 b and flows downstream in flowdirection 30. During this time, the second liquid/third liquid interfaceremains static. Labeled reagent present in the second liquid binds tocomplex formed at reaction zone 20, if present.

When all second liquid is drained from middle reservoir 32 b, the secondliquid/third liquid interface begins to move downstream in flowdirection 30 as third liquid from upstream reservoir 32 c drains downthrough liquid receiving zone 34 c and flows downstream in flowdirection 30. Signal producing component present in the third liquidreacts with the labeled reagent bound to complex, to generate anobservable signal

As discussed in the introduction, efforts have previously been made toimplement methods that resemble the method of the present invention buthave failed. In the prior art attempts, an interface between two liquidsis formed but no standing columns of liquid is produced in a reservoir.Rather, liquids invariably leak from various locations on the capillaryflow matrix, generally anywhere the capillary flow matrix makes contactwith a physical object thus forming an alternate capillary pathway. Forexample, in a multireservoir lateral flow capillary device such asdisclosed in U.S. Pat. No. 4,981,786 leakage occurs along supportcomponents on which the capillary flow matrix rests, along laterallydisposed support components that hold the capillary flow in place and upthe contact points of reservoirs with the capillary flow matrix,producing a flow of liquid on the top surface of the capillary flowmatrix.

Thus, in contrast to the methods and lateral flow capillary devicesknown in the art, the teachings of the present invention allowperformance of multistep reactions using a lateral flow capillary devicewhere each step is performed with a relatively precise amount of reagentfor a relatively precise duration. Since leakage is prevented and sincethe duration of a reaction step is accurately determined by the volumeof the different liquids added, many different multistep experiments canbe performed to yield repeatable results. Further, as the volume ofliquid is the primary determinant of duration of a given step, theduration of a given step is easily modified if required, allowingperformance of kinetic experiments.

Although, not wishing to be held to any one theory, it is believed thatthe reason for the failure of earlier attempts and the success of theinventor in successfully implementing the concept of liquid columns influid communication with a capillary flow matrix for use in performingmultistep reactions is related to the forces acting on a liquid inside acapillary flow matrix and the elimination of potential alternatecapillary pathways.

Water potential T of the liquid is the potential energy of water in agiven volume (mass) and determines flow direction from a volume of ahigher water potential to a volume with a lower water potential. Thetotal water potential Ψ of a volume of water is the sum of fourcomponent potentials: gravitational (Ψg), matrix (Ψm), osmotic (Ψs), andpressure (Ψp). Gravitational potential depends on the position of thewater in a gravitational field. Matrix potential depends on theadsorptive forces binding water to a matrix. Osmotic potential dependson the concentration of dissolved substance in the water (e.g., asolution having a high salt concentration has a negative value).Pressure potential depends on the hydrostatic or pneumatic pressure onthe water. Matrix potential is affected by both matrix and liquidproperties. Matrix potential is affected by the attraction of the liquidto the matrix (hydrophilicity) and the surface area of the cavities inthe matrix.

In a single reservoir lateral flow capillary device, the force acting ona liquid inside the capillary flow matrix includes the force applied bya single column of liquid in a reservoir contributing to Ψp. Theattraction of the liquid molecules to the internal surfaces of thecapillary flow matrix (Ψm) are sufficient to prevent leakage of liquidalong alternate capillary paths formed, for example, where some physicalobject contacts the capillary flow matrix.

In a multireservoir lateral flow capillary device, described herein theforce acting on a liquid inside the capillary flow matrix includes theforce applied by two standing columns of liquid in two reservoirs bothcontributing to Ψp. In such cases, Ψm that is a measure of theattraction between the liquid molecules and the internal surfaces of thecapillary flow matrix are insufficient to prevent leakage of liquidalong alternate capillary flow paths.

In embodiments of a lateral flow capillary device of the presentinvention, contact points are eliminated except at the rims of thereservoirs and, if present, at the oppositely disposed supportingcomponents, which are pressed into the capillary flow matrix. Thepressing of these components locally compresses the matrix and the porestherein, reducing the volume of the matrix proximal to these componentsbut not changing the total internal surface area. Such pressingincreases the capillary flow matrix/liquid interaction energy per unitvolume in the vicinity of the components and therefore increases Ψm.This increased energy is apparently sufficient to compensate for theincreased force applied by the two columns of liquid. In summary,pressing of the matrix increases the binding energy in the vicinity ofthe contact points with the rims or supporting components, reducing thetendency to leak across the alternative capillary flow path formed atthe contact point.

In embodiments of the present invention, a given interface creating zoneis relatively wide relative to flanking liquid receiving zones. Anadvantage of such embodiments is that the size of the interface creatingzone means that liquid added to one of the liquid receiving zonesrequires a significant period of time to travel through the interfacecreating zone before arriving at the neighboring liquid receiving zone.This allows addition of liquids to different reservoirs to be sequentialand to be performed under more difficult (e.g., non laboratory)conditions, and even by less skilled operators. An additional advantageof a relatively wide interface creation zone is to compensate fordifferent absorption rates of different liquids added. For example, therate of entry of a viscous or hydrophobic liquid is relatively slow. Arelatively wide interface creation zone prevents faster matrix enteringliquids from travelling to a liquid receiving zone where a viscous orhydrophobic liquid is added, allowing the viscous or hydrophobic liquidto enter the matrix and create a sharp, well-defined liquid-liquidinterface.

Further, it has been found that the relatively long interface creatingzones leads to the formation of more sharply defined interfaces that aresubstantially perpendicular to the flow direction.

Many materials used as capillary flow matrices swell upon contact withwater. In lateral flow capillary devices where the liquid inducedswelling of the interface creation zone is constrained, for example whenpressure is applied thereto from above, the swelling may be inhomogenousand cracks may form in the matrix, locally modifying the capillaryproperties of the matrix. If the interface forms in a volume where thecapillary properties are changed, the interface formed between liquidsmay be indefinite and not clear, leading to mixing of the two liquids ofthe interface and concomitant negative effects. In embodiments of thepresent invention, liquid induced swelling of the interface creationzone is unconstrained.

In embodiments of the present invention, the first liquid and secondliquid are added substantially simultaneously to a respective reservoir.

In embodiments of the present invention, the first liquid and secondliquid are added sequentially. When the liquids are added sequentially,the order in which the liquids is added it is of little significance, aslong as a subsequent liquid is added to a respective reservoir before anearlier added liquid migrates into the liquid receiving zone of thereservoir of the subsequently added liquid.

As noted above, the method of the present invention as described aboveis not directed to the transport of liquids through a capillary flowmatrix, but rather allows the performance of multistep reactionsespecially multistep specific binding assays, where each of the twoliquids initiates and performs a different step of a multistep reaction,for example transport of a reagent. As is clear to one skilled in theart upon perusal of the description herein, the volume of any givenliquid determines to a large degree the duration of a respective step.

Depending on the purpose for which lateral flow capillary device 24 isdesigned, reaction zone 20 comprises at least one capturing entity(e.g., a member of a specific binding pair) configured to capture amaterial (e.g., an analyte or a product of a reaction involving theanalyte) flowing through the capillary flow matrix. In embodiments ofthe present invention, the reaction zone is in a liquid receiving zoneof a respective reservoir.

In embodiments of the present invention, a lateral flow capillary deviceis provided with one or more reagents pre-loaded onto the capillary flowmatrix. Such preloading of reagents is known in the art of lateral flowcapillary devices, for example by drying reagents onto the matrix, forexample by freeze drying, spray drying, dispensing and air drying.

In brief, a reagent is loaded onto the capillary flow matrix in such away that a liquid added through a specific reservoir will interact withthe reagent. In embodiments, at least one pre-loaded reagent isconfigured to react with an added analyte to produce a reaction productthat is subsequently transported downstream along the capillary flowmatrix. In embodiments, at least one pre-loaded reagent is configured tobe solubilized by an added liquid and to be subsequently transporteddownstream along the capillary flow matrix. In embodiments, a preloadedreagent is located substantially in a liquid receiving zone. Inembodiments, a preloaded reagent is located in the vicinity of a liquidreceiving zone, specifically in an adjacent interface creation zone.

Embodiments of the present invention allow material to be preloaded bothupstream and downstream of a given liquid receiving zone, allowingmaterial to be preloaded in a relatively large region of matrix usingsimple methods, for example by spray-drying. Generally, materialpreloaded downstream from the most downstream liquid receiving zone ispreloaded at any distance from the liquid receiving zone, generally (butnot necessarily) between the liquid receiving zone and a reaction zone.Material preloaded in the vicinity of a most upstream liquid receivingzone is generally loaded downstream from the liquid receiving zone sothat all the material will be used when liquid is introduced into themost upstream liquid receiving zone. Other preloaded material isgenerally preloaded either in the liquid receiving zone or somewhatupstream or somewhat downstream from the liquid receiving zone, forexample up to about 30% of the length of the adjacent interface creationzone.

In embodiments of the present invention, opposite the rims of the liquidconduits of the reservoirs the matrix is attached to a substantiallyimpermeable backing material to avoid leakage of liquids from thecapillary flow matrix.

In FIG. 4 is depicted an embodiment of a lateral flow capillary deviceof the present invention, 46.

In lateral flow capillary device 46, capillary flow matrix 18 isattached to a substantially impermeable backing 48. Together, capillaryflow matrix 18 and impermeable backing 48 constitute a strip that restson plateau 50, where backing 48 contacts plateau 50. lateral flowcapillary device 46 is provided with four reservoirs 32 a, 32 b, 32 cand 32 d with respective rims 36 a, 36 b, 36 c and 36 d that press theupper surface of capillary flow matrix 18. Plateau 50 is disposedopposite each rim 36 of each reservoir 32 and thus constitutes asupporting component supporting matrix 18 against the pressing of rims36.

Lateral flow capillary device 46 is configured for the simpleperformance of four-step reactions.

A first reagent 52 is preloaded in liquid receiving zone 34 a, firstreagent 52 being a nutrient configured to cause living cells contactingfirst reagent 52 to express certain proteins on external membranes.

A second reagent 54 is preloaded to an area of capillary matrix 18between liquid receiving zones 34 b and 34 c, second reagent 54 being atoxin.

A third reagent 56 is preloaded to liquid receiving zone 34 d, thirdreagent 56 being an indicator that binds to the certain protein.

Reaction zone 20 includes a capture entity configured to immobilizecells.

The use of lateral flow capillary device 46 is substantially similar tothe use of lateral flow capillary device 24 as described above and isclear to one skilled in the art upon perusal of the description herein.

A first liquid sample, including living cells is placed in reservoir 32a, a second liquid placed in reservoir 32 b, a third liquid placed inreservoir 32 c and a fourth liquid placed in reservoir 32 d,sequentially or simultaneously. Three liquid interfaces are formed inthe interface creation zones 35 a, 35 b, 35 c, the interface 42 a, 42 b,42 c. Standing columns of liquid are produced in reservoirs 32 b, 32 cand 32 d. When the cell-containing first liquid sample contacts firstreagent in liquid receiving zone 34 a, the cells begin to produce thespecific metabolite. When the liquid sample reach reaction zone 20 thecells are immobilized.

When first liquid sample in reservoir 32 a is exhausted, second liquidin reservoir 32 b begins to flow through capillary flow matrix 18,transporting waste and other non-bound material away from reaction zone20 towards liquid drain 23.

When second liquid in reservoir 32 b is exhausted, third liquid inreservoir 32 d begins to flow through capillary flow matrix 18, carryingthe second reagent 54. Second reagent 54 is transported to reaction zone20, killing immobilized cells.

When third liquid in reservoir 32 c is exhausted, fourth liquid inreservoir 32 d begins to flow through capillary flow matrix 18, carryingthe third reagent 56. Third reagent 56 is transported to reaction zone20, producing a visible signal on cells that expressed the certainproteins on external membranes.

Using a plurality of lateral flow capillary devices 46, substantiallyidentical experiments are performed where only the amount of secondliquid added to reservoir 32 b is varied, thus varying the time betweenexposure of cells to first reagent 52 and killing of the cells withexposure to second reagent 54. In such a way, the kinetics of proteinexpression is studied.

In embodiments of the invention is used for applications other thandiagnostic applications including biomolecule extraction or synthesis,for example, concentration of nucleic acids in a sample. Nucleic acidabsorption particles are attached to a reaction zone of a capillary flowmatrix and a sample containing nucleic acids is added under bindingconditions resulting in concentration of nucleic acid from the sample.In embodiments the concentration step is followed by washing, elutionand/or analysis steps.

Methods of Manufacture of a Lateral Flow Capillary Device of the PresentInvention

In general, manufacture and assembly of a lateral flow capillary deviceof the present invention is well within the ability of one skilled inthe art upon perusal of the description and figures herein using anysuitable method with which one skilled in the art is well acquainted.Suitable methods include methods that employ one or more techniquesincluding but not limited to welding, casting, embossing, etching,free-form manufacture, injection-molding, microetching, micromachining,microplating, molding, spin coating, lithography or photo-lithography.

In an aspect of the present invention, a device and a kit are providedallowing the simple and cheap preparation of a custom lateral flowcapillary device in accordance with the teachings of the presentinvention.

A device 60 of the present invention useful for the preparation oflateral flow capillary device is depicted in FIG. 5A. Device 60comprises a first component 62 and a second component 64, connectedthrough protruding extensions 66 and 68 by a hinge 70.

First component 62 includes a reservoir 32 having one wall configured tohold liquids. The lowest area of reservoir 32 is a non-capillary opening63 defining a hollow conduit with a rim 36. In embodiments rim 36 isrelatively wide, at least about 0.5 mm or even at least about 1 mm. Inembodiments, rim 36 is substantially planar. To be non-capillary, inembodiments non-capillary opening 63 is relatively large, in embodimentshaving a cross-sectional area of at least about 1 mm², of at least about3 mm² or even of at least about 7 mm² Opposite extension 66 and alsoprotruding from reservoir 32 is protrusion 72.

Second component 64 includes a body 74 with a counter support platform76 at the top end of body 74. Opposite extension 68 and also protrudingfrom body 74 is protrusion 78.

Protrusions 72 and 78 are configured to mutually engage, interlocking(for example by “snapping together”) so as to hold rim 36 and countersupport platform 76 substantially parallel spaced apart at somepredetermined distance.

In device 60, first component 62 is provided with two extensions 66 and72 to engage two extensions 68 and 78 of second component 64. Inembodiments, a first component and/or a second component are providedwith only one extension, or with more than two such extensions each.

In device 60, extensions 66 and 68 are formed as one piece to definehinge 70. In embodiments of the present invention, extensions areconfigured to be reversibly or irreversibly engaged, when engageddefining a hinge. In embodiments of the present invention, extensionsare configured to be reversibly or irreversibly engaged, when engagednot defining a hinge.

The use of a device of the present invention such as device 60 in theframework of a kit of the present invention, is depicted in from the topin FIG. 5B and from the side in FIG. 5C. In FIGS. 5B and 5C, a kit ofthe present invention comprising two devices 60 and a strip of plasticbacked glass fiber as a bibulous capillary flow matrix 18 is depicted inan assembled state, together constituting an embodiment of the device ofthe present invention.

For assembly, capillary flow matrix 18 of an appropriate thickness iscut to size and two devices 60 are closed over an appropriate area ofcapillary flow matrix 18. The thickness of capillary flow matrix 18 andthe design of devices 60 is such that, when extensions 72 and 78 aremutually engaged, capillary flow matrix 18 is clamped between rim 36 andcounter support platform 76 In such a state, non-capillary openings 63defines a liquid receiving zone. Further, rim 36 presses into capillaryflow matrix 18, in accordance with the teachings of the presentinvention.

As is clear to one skilled in the art upon perusal of the description alateral flow capillary device, including a lateral flow capillary deviceof the present invention is easily custom built and modified with theuse of embodiments of devices of the present invention and embodimentsof kits of the present invention. For example, application of desiredreagents to define a reaction zone or to preload a reagent onto acapillary matrix is simple to achieve.

In the art, for example in U.S. Pat. No. 4,981,786, is taught theintroduction of sample or substrate in a downstream reservoir andaddition of a carrier liquid in an upstream reservoir to transport anreagent located on a capillary flow matrix downstream to contact thesample or substrate. In an aspect of the present invention is taught amethod where a sample is also used as a carrier liquid.

In the method, a lateral flow capillary device substantially asdescribed above is used including: a first liquid receiving zone on thecapillary flow matrix in fluid communication with a first reservoir, asecond liquid receiving zone on the capillary flow matrix in fluidcommunication with a second reservoir upstream of the first reservoir, afirst reagent preloaded inside the first reservoir and/or at a locationon the capillary flow matrix in proximity to the first liquid receivingzone or downstream therefrom; a second reagent preloaded at a locationinside the second reservoir and/or on the capillary flow matrix inproximity to the second liquid receiving zone.

A first amount of a liquid (e.g., the sample) is added to the firstreservoir so that the liquid flows into the capillary flow matrixthrough the first liquid receiving zone to contact the first reagent,e.g., to react with the first reagent or to solubilize the firstreagent.

A second amount of a liquid (either the same or different) is added tothe second reservoir so that the liquid flows into the capillary flowmatrix through the second liquid receiving zone to contact the secondreagent. The second amount of liquid is added before, after orsubstantially simultaneously with the addition of the first amount ofliquid.

In accordance with the teachings of the present invention, when thefirst amount and the second amounts are such that liquid substantiallyremains in the first and second reservoirs respectively, a staticinterface is formed between the liquid contacting the first reagent andthe liquid contacting the second reagent in the interface creation zonebetween the two liquid receiving zones. In accordance with the teachingsof the present invention as described above, the interface moves onlysubsequent to exhaustion of liquid from the first reservoir. Inembodiments, there is a reaction zone downstream of the first liquidreceiving zone on the capillary flow matrix. In embodiments, there is areaction zone in the first liquid receiving zone. As is clear to oneskilled, the advantage of this method is that an assay is made verysimple.

When the liquid added to the first reservoir is the same as that addedto the second reservoir, only one liquid is added (for example through asingle port in communication with a number of liquid receiving zones) asin a single reservoir lateral flow capillary device but performs amultistep reaction with all the advantages thereof. This reduces thenumber of steps required to perform an otherwise complex multistepreaction.

EXPERIMENTAL

Reference is now made to the following examples, which together with theabove descriptions, demonstrate the invention in a non limiting fashion.

Experiment 1 Preparation of an Embodiment of a Lateral Flow CapillaryDevice of the Present Invention

A lateral flow capillary device such as lateral flow capillary device 80depicted in FIGS. 6A, 6B and 6C was prepared.

A lower housing part 82 and an upper housing part 84 configured to snaptogether to form a closed shell holding a capillary flow matrix 18 andtwo liquid drains 86 and 88 were fashioned by injection molding of ABS(acrylonitrile-butadiene-styrene copolymer) plastic. Lower housing 82was substantially a lidless box having a bottom with a plateau portion50 and a recessed portion 90. Upper housing 84 was substantially a lidfor lower housing 82 and was provided with three reservoirs A, B and Cincluding circular rims 36 a, 36 b, 36 c, a observation window 22, andfour drain-pressing protrusions 92.

Capillary flow matrix 18, substantially a 50 mm×32 mm porous membrane ofGF grade 161 glass fiber from Ahlstrom Corporation (Helsinki, Finland)attached to 55 m×32 mm×0.5 mm thick adhesive coated plastic backing(high-impact polystyrene coated with an adhesive LH-50 from AdvancedMicrodevices Pvt. Ltd. Ambala Cantt, India) so that the upstream end 26of capillary flow matrix 18 was flush with an end of backing 48 and 5 mmof adhesive-coated backing protruded from upstream end 93 of backing 48.

A test line 20 a and a control line 20 b, constituting a reaction zone20 were applied as two parallel lines of spots of materials to capillaryflow matrix 18 using a laboratory pipette see FIG. 6B.

Test line 20 a was applied as a line of spots produced by applying 1microliter of 0.7 mg/mL Goat anti Rabbit Ab (Jackson ImmuonResearchlaboratories Inc. 111-005-046) in 0.1 M phosphate buffer (pH 6.8) and 2%trehalose solution 36 mm from the upstream end of capillary flow matrix18.

Control line 20 b was applied as a line of spots produced by applying 1microliter of Rabbit Ab 0.1 mg/ml Rabbit anti calf Alkaline Phosphates(Biogenesis 0300-1024) and 0.4 mg/ml Rabbit IgG I 5006 (Sigma-Aldrich,St. Louis, Mo., USA) in 0.1 M phosphate buffer (pH 6.8) and 2% trehalosesolution 42 mm from the upstream end of capillary flow matrix 18.

After application of the spots, capillary flow matrix 18 was dried at37° C. for 15 minutes, treated with a solution of 0.5% gelatin, 2.5%Bacto-Tryptone, 1% trehalose in PBS and then dried at 37° C. for anadditional 2 hours.

Two liquid drains 86 and 88 were prepared of highly absorbent purecellulose paper with a very high flow rate (190 mm/30 min) Chr. 17(Whatman). Upper liquid drain 86 was 32 mm×36 mm and attached to theadhesive of protruding upstream end 93 of backing 48 abutting capillaryflow matrix 18 so as to ensure fluid communication therewith. Lowerliquid drain 88 was 7.8 mm×83 mm.

As depicted in FIG. 6C, for assembly of lateral flow capillary device80, lower liquid drain 88 was laid in recess 90 of lower housing 82,capillary flow matrix 18 together with upper liquid drain 86 were placedon plateau 50 of lower housing 82 with backing 48 making contact withplateau 50. Upper housing 84 was pressed into place to engage and snaptogether with lower housing 82 so that rims 36 of reservoirs A, B and Cpressed into capillary flow matrix 18 to define liquid receiving zones34 a, 34 b and 34 c and so that drain pressing protrusions 92 pressedthe end of upper liquid drain 86 to contact lower liquid drain 88.

Experiment 2 Use of a Lateral Flow Capillary Device to Study EnzymaticReaction

Three lyophilized reagents were prepared:

Reagent A

11-dehydro-TxB2-antiserum reagent—150 ul Rabbit anti 11-dehydro-TXB,2999-044 (Assay Designs, Inc.) diluted 1:15000 in 1% BSA, 0.25%TWEEN-20, 0.1 mM ZnCl₂, 1 mM MgCl₂ in PBS buffer pH 7.4 was placed in avial, cooled to −80° C. and lyophilized for 24 hours.

Reagent B

Enzyme labeled 11-dehydro-TxB2 conjugate—150 ul 11-dehydro-TxB2-AlkalinePhosphatase conjugate, 1:80 DCC (Assay Designs, Inc.) diluted 1:30,000in 1% BSA, 0.25% TWEEN-20, 0.1 mM ZnCl₂, 1 mM MgCl₂ in PBS buffer pH 7.4was placed in a vial, cooled to −80° C. and lyophilized for 24 hours.

Reagent C

AP substrate—BCIP/NBT prepared according to manufacturer instruction:stock preparation—1 tablet BCIP, B0274 (Sigma-Aldrich, St. Louis, Mo.USA) dissolved in 1 ml DMF, 1 tablet NBT, N55141 (Sigma-Aldrich, St.Louis, Mo., USA) dissolved in 1 ml water. 300 ul of the combinedsolution, 33 ul BCIP, 333 ul NBT stock solutions, in 10 ml 0.1M Trisbuffer pH 9.7, were placed in a vial, cooled to −80° C. and lyophilizedfor 24 hours.

Three lateral flow capillary devices were prepared:

A first lateral flow capillary device was prepared substantially asdescribed above with dry PBS buffer placed in reservoir A, reagent Bplaced in reservoir B and reagent C placed in reservoir C.

A second lateral flow capillary device was prepared substantially asdescribed above with reagent A placed in reservoir A, dry PBS solutionplaced in reservoir B and reagent C placed in reservoir C.

A third lateral flow capillary device was prepared substantially asdescribed above with reagent A placed in reservoir A, reagent B placedin reservoir B and reagent C placed in reservoir C.

To each of the three lateral flow capillary devices was added doubledistilled water: 150 microliter in reservoir A, 150 microliter inreservoir B and 300 microliter in reservoir C, one reservoir after theother. A standing column of liquid was seen in each reservoir and, asdescribed above in accordance with the teachings of the presentinvention, the liquid drained first from reservoir A, then fromreservoir B and finally from reservoir C. When all liquid drained awayfrom reservoir C, the enzymatic reaction was stopped by the addition ofa 120 ul 0.25M sulfuric acid stop solution to reservoir C.

The colors of the test lines and control lines were measured using aPART Pro Reader (LRE Technology Partner GmbH) and depicted in FIG. 7.

As seen in FIG. 7, in the first lateral flow capillary device, no colorwas observed at the test line and color was observed at the controlline; in the second lateral flow capillary device, color was observed atneither the test line nor at the control line; and in the third lateralflow capillary device, color was observed at both the test line and atthe control line.

The results indicate that the lateral flow capillary devices operated asexpected.

Experiment 3 TxB2 Detection for Comparing a Multireservoir Lateral FlowCapillary Device with a Single Reservoir Lateral Flow Capillary Devicefor Performing a Multistep Reaction

Two lateral flow capillary devices A and B were prepared substantiallyas described above in Experiment 1 with a 50 mm×32 mm capillary flowmatrix and two capillary flow reactors C and D were prepared with ashorter 40 mm×32 mm capillary flow matrix and assembled so that only rim34 a of reservoir a was in contact with capillary flow matrix 18.

Five reaction liquids were prepared:

1. A diluent solution of 1% BSA, 0.25% TWEEN-20, 0.1 mM ZnCl₂, 1 mMMgCl₂ in PBS buffer pH 7.4;

2. Reagent A of Rabbit anti 11-dehydro-TXB2 (Assay Designs, Inc.999-044) diluted 1:15,000 in 1% BSA, 0.25% TWEEN-20, 0.1 mM ZnCl₂, 1 mMMgCl₂ in PBS buffer pH 7.4;

3. Reagent B of 11-dehydro-TxB2—Alkaline Phosphatase conjugate (AssayDesigns, Inc. 1:80 DCC) diluted 1:30,000 in 1% BSA, 0.25% TWEEN-20, 0.1mM ZnCl₂, 1 mM MgCl₂ in PBS buffer pH 7.4;

4. Reagent C of BCIP/NBT prepared according to manufacturer instruction:stock preparation—1 tablet BCIP (Sigma-Aldrich B0274) dissolving in 1 mlDMF, 1 tablet NBT (Sigma-Aldrich N55141) dissolving in 1 ml water, finalsolution: 33 ul BCIP, 333 ul NBT stock solutions, in 10 ml 0.1M Trisbuffer pH 9.7; and

5. Reagent D, a stop solution of 0.25M sulfuric acid.

Use of Multireservoir Lateral Flow Capillary Devices

150 ul of reagent A, 150 ul of reagent B and 300 ul of reagent C weresimultaneously added to reservoirs A, B, and C respectively of lateralflow capillary device A.

150 ul of diluent solution, 150 ul of reagent B and 300 ul of reagent Cwere simultaneously added to reservoirs A, B, and C respectively oflateral flow capillary device B.

After complete sequential draining of all three solutions in the orderA, B and C, 120 ul reagent D was added to reservoir C.

The colors of the test lines and control lines were measured using aPART Pro Reader (LRE Technology Partner GmbH) and depicted in FIGS. 8Aand 8B.

Use of Single Reservoir Lateral Flow Capillary Devices

To reservoir A of lateral flow capillary device C were added one afterthe other 150 ul of reagent A, 150 ul of reagent B, 300 ul of reagent Cand 120 ul of reagent D, each succeeding liquid only after the previousliquid had completely drained away from the reservoir.

To reservoir A of lateral flow capillary device D were added one afterthe other 150 ul of diluent solution, 150 ul of reagent B and 300 ul ofreagent C and 120 ul of reagent D, each succeeding liquid only after theprevious liquid had completely drained away from the reservoir.

The colors of the test lines and control lines were measured using aPART Pro Reader (LRE Technology Partner GmbH) and depicted in FIGS. 8Cand 8D.

From comparing FIGS. 8A and 8C and FIGS. 8B and 8D it is seen that theresults obtained using a multireservoir lateral flow capillary flowdevice when adding all reagents at the beginning of the experiment aresubstantially the same as the results obtained using a single reservoirlateral capillary flow device when adding the reagents during theexperiment.

The time duration of each reservoir draining was measured and the flowrat was calculated (minutes for 100 ul liquid to travel 1 cm through thecapillary flow matrix results shown in Table 1:

TABLE 1 Flow [minutes for 100 ul to travel 1 cm] Liquid Reservoir deviceA device B device C device D A A 0:00:57 0:00:57 B B 0:01:10 0:01:16 C C0:01:08 0:01:10 D C 0:01:16 0:01:19 A A 0:00:55 0:00:54 B A 0:01:140:01:10 C A 0:01:12 0:01:08 D A 0:01:16 0:01:10

Experiment 4 Calibration Curve for 11-Dehydro-TxB2-Competition Assay

Five lateral flow capillary devices substantially as described above inexperiment 1

Five reagent liquids were prepared:

1. A diluent solution of 1% BSA, 0.25% TWEEN-20, 0.1 mM ZnCl₂, 1 mMMgCl₂ in PBS buffer pH 7.4;

2. Reagent A of Rabbit anti 11-dehydro-TXB2 (Assay Designs, Inc.999-044) diluted 1:1,500 in 1% BSA, 0.25% TWEEN-20, 0.1 mM ZnCl₂, 1 mMMgCl₂ in PBS buffer pH 7.4;

3. Reagent B of 11-dehydro-TxB2—Alkaline Phosphatase conjugate (AssayDesigns, Inc. 1:80 DCC) diluted 1:3,000 in 1% BSA, 0.25% TWEEN-20, 0.1mM ZnCl₂, 1 mM MgCl₂ in PBS buffer pH 7.4;

4. Reagent C of BCIP/NBT was prepared according to manufacturerinstruction: stock preparation—1 tablet BCIP (Sigma-Aldrich B0274)dissolving in 1 ml DMF, 1 tablet NBT (Sigma-Aldrich N55141) dissolvingin 1 ml water. Final solution: 33 ul BCIP, 333 ul NBT stock solutions,in 10 ml 0.1M Tris buffer pH 9.7; and

5. Reagent D, a stop solution of 0.25M sulfuric acid.

Five sample solutions were prepared containing 5, 1, 0.2, 0.04, 0 ng/mlof 11-dehydro-TxB2 (80-0735) analyte from Assay Designs, Inc dissolvedin 1% BSA, 0.25% TWEEN-20, 0.1 mM ZnCl₂, 1 mM MgCl₂ in PBS buffersolution pH 7.4.

To the first lateral flow capillary device, 150 ul of a mixture of 15 ulreagent A and 135 ul sample containing 5 ng/ml analyte, 150 ul of amixture of 15 ul reagent B and 135 ul sample containing 5 ng/ml analyteand 300 ul of reagent C were added to reservoirs A, B, and Crespectively.

To the second lateral flow capillary device, 150 ul of a mixture of 15ul reagent A and 135 ul sample containing 1 ng/ml analyte, 150 ul of amixture of 15 ul reagent B and 135 ul sample containing 1 ng/ml analyteand 300 ul of reagent C were added to reservoirs A, B, and C.

To the third lateral flow capillary device, 150 ul of a mixture of 15 ulreagent A and 135 ul sample containing 0.2 ng/ml analyte, 150 ul of amixture of 15 ul reagent B and 135 ul sample containing 0.2 ng/mlanalyte and 300 ul of reagent C were added to reservoirs A, B, and C.

To the fourth lateral flow capillary device, 150 ul of a mixture of 15ul reagent A and 135 ul sample containing 0.04 ng/ml analyte, 150 ul ofa mixture of 15 ul reagent B and 135 ul sample containing 0.04 ng/mlanalyte and 300 ul of reagent C were added to reservoirs A, B, and Crespectively.

To the fifth lateral flow capillary device, 150 ul of a mixture of 15 ulreagent A and 135 ul sample containing 0 ng/ml analyte, 150 ul of amixture of 15 ul reagent B and 135 ul sample containing 0 ng/ml analyteand 300 ul of reagent C were added to reservoirs A, B, and Crespectively.

After complete draining of all three reservoirs in the order A, B and Cin accordance with the teachings of the present invention, 120 ulreagent D was added to reservoir C of each of the lateral flow capillarydevice.

The colors of the test lines and control lines were measured using aPART Pro Reader (LRE Technology Partner GmbH) and the bound level of thelabeled analyte at each analyte concentration was calculated as theratio b/b0 between the reflection at each concentration 5, 1, 0.2, 0.04ng/ml (b) and the reflection at 0 ng/ml (b0). A calibration curve wasmade by plotting the results, FIG. 9.

Experiment 5 Quantitative Determination of 11-Dehydro-TxB2 in Urine

Three lateral flow capillary devices substantially similar to the thirdlateral flow capillary device described in Experiment 2 were preparedwith lyophilized reagent A in reservoir A, lyophilized reagent B inreservoir B and lyophilized reagent C in reservoir C.

To each of the three lateral flow capillary devices was added: 150 ulurine sample to reservoir A, 150 ul of the same urine sample toreservoir B and 300 ul double distilled water to reservoir C. Aftercomplete sequential draining of reservoirs A, B, and C in accordancewith the teachings of the present invention, 120 ul stop solution D wasadded to reservoir C.

The colors of the test lines and control lines were measured using aPART Pro Reader (LRE Technology Partner GmbH) and the concentration of11-dehydro-TxB2 analyte in each urine sample determined with referenceto the calibration curve of FIG. 9. The first urine sample wasdetermined to contain 5251 pg/mL, the second urine sample 907 pg/ml andthe third urine sample 540 pg/ml 11-dehydro-TxB2.

Experiment 6 Sequential Liquid Flow in a Lateral Flow Capillary Device

A lateral flow capillary device E was prepared substantially asdescribed above in Experiment 1 with a 50 mm×32 mm capillary flowmatrix. A lateral flow capillary device F were prepared with a shorter40 mm×32 mm capillary flow matrix and assembled so that only rim 36 a ofreservoir A was in contact with capillary flow matrix 18. The capillaryflow matrices of both lateral flow capillary devices E and F were devoidof reaction zones and only treated with a solution of 0.5% gelatin, 2.5%Bacto-Tryptone, 1% trehalose in PBS.

A number of reaction liquids, diluent solution, reagent A (yellow),reagent B (blue) and reagent C (red) were prepared as described inExperiment 3

Use of Multireservoir Lateral Flow Capillary Device

150 ul of reagent A, 150 ul of reagent B and 300 ul of reagent C wereadded, one after the other, to reservoirs A, B, and C respectively oflateral flow capillary device E. Sequential draining of reservoirs A, B,and C in accordance with the teachings of the present invention wasobserved with a sharp interface that was observed to move in accordancewith the teachings of the present invention.

Use of Single Reservoir Lateral Flow Capillary Device

To reservoir A of lateral flow capillary device F were added one afterthe other 150 ul of reagent A, 150 ul of reagent B and 300 ul of reagentC each succeeding liquid only after the previous liquid had completelydrained away.

The draining time for each reservoir was measured and listed in Table 2:

TABLE 2 Draining time Liquid Reservoir device E device F A A 0:3:07 B B0:10:01 C C 0:25:20 A A 0:03:03 B A 0:08:14 C A 0:18:13

Experiment 7 Detection of HIV 1 Antibodies in Blood Serum Sample aLateral Flow Capillary Device was Prepared Substantially as DescribedAbove in

Experiment 1 with a control line 20 b as described in Experiment 1 butwith a test line 20 a applied as a line of spots produced by applying 1microliter of 0.7 mg/mL HIV 1 recombinant protein antigen HIV-101(ProSpec-Tany TechnoGene LTD) in 0.1 M phosphate buffer (pH 6.8) and 2%trehalose solution 36 mm from the upstream end of capillary flow matrix18.

In reservoir A was placed a lyophilized (as described above) solution of1 mg/ml biotinylated synthetic gp41 and gp120 peptides diluted in 1%BSA, 1% fetal bovine serum, 0.5% TWEEN-20 in PBS buffer pH 7.4.

In reservoir B was placed a lyophilized (as described above) solution ofStreptavidin-Alkaline Phosphatase conjugate (Jackson ImmuonResearchlaboratories Inc. 003-050-083) diluted in 1% BSA, 0.5% TWEEN-20, 0.1 mMZnCl₂, 1 mM MgCl₂ in PBS buffer pH 7.4.

In reservoir C was placed a lyophilized (as described above) solution ofBCIP/NBT prepared according to the manufacturer instructions stockpreparation—1 tablet BCIP (Sigma-Aldrich B0274) dissolving in 1 ml DMF,1 tablet NBT (Sigma-Aldrich N55141) dissolving in 1 ml water. A finalsolution: 33 ul BCIP, 333 ul NBT stock solutions, in 10 ml 0.1M Trisbuffer pH 9.7.

150 ul of a serum sample, 150 ul of a serum sample and 300 ul of doubledistilled water were added, one after the other, to reservoirs A, B, andC respectively of the lateral flow capillary device. Sequential drainingof reservoirs A, B, and C in accordance with the teachings of thepresent invention was observed. After the liquid in reservoir Ccompletely drained away 120 ul reagent D (0.25 M sulfuric acid stopsolution) was added to reservoir C.

The appearance of two colored dotted lines, one at the test line and theother at the control line, indicated the presence of antibodies for HIV1 in the serum sample.

Experiment 8 Detection of Hepatitis B Surface Antigen in Blood SerumSample Using a Two Reservoir Lateral Flow Capillary Device

A lateral flow capillary device was prepared substantially as describedabove in Experiment 1 excepting that the capillary flow matrix was 45mm×32 mm and the lower liquid drain was 7.8 mm×73 mm and with a controlline 20 b as described in Experiment 1 but with a test line 20 a appliedas a line of spots produced by applying 1 microliter of 0.7 mg/mL mousemonoclonal anti-HBsAg antibody (Fitzgerald Industries International,Inc. 10-H05) in 0.1 M phosphate buffer (pH 6.8) and 2% trehalosesolution. When assembled in the housing, the rims 34 a and 34 b ofreservoirs A and B were in contact with capillary flow matrix 18 but therim 36 c of reservoir C was not in contact with the capillary flowmatrix 18.

In reservoir A was placed a lyophilized (as described above) solution ofrabbit anti-HBsAg Alkaline Phosphatase conjugate diluted in 1% BSA, 1%fetal bovine serum, 0.5% TWEEN-20, 0.1 mM ZnCl₂, 1 mM MgCl₂ in PBSbuffer pH 7.4.

In reservoir B was placed a lyophilized (as described above) solution ofBCIP/NBT prepared according to the manufacturer instructions stockpreparation—1 tablet BCIP (Sigma-Aldrich B0274) dissolving in 1 ml DMF,1 tablet NBT (Sigma-Aldrich N55141) dissolving in 1 ml water. A finalsolution: 33 ul BCIP, 333 ul NBT stock solutions, in 10 ml 0.1M Trisbuffer pH 9.7.

300 ul of a serum sample and 300 ul of double distilled water wereadded, one after the other, to reservoirs A and B respectively of thelateral flow capillary device. Sequential draining of reservoirs A and Bin accordance with the teachings of the present invention was observed.After the liquid in reservoir B completely drained away 120 ul 0.25 Msulfuric acid stop solution was added to reservoir B.

The appearance of two colored dotted lines, one at the test line and theother at the control line, indicated the presence of Hepatitis B SurfaceAntigen in the serum sample.

Experiment 9 Detection of Fluorescent Signal—High Volume Samples

Four lateral flow capillary devices were prepared substantially asdescribed in Experiment 1 where reaction zone 20 included only a testline 20 a but no control line.

A reagent H was prepared of Rabbit anti mouse-cy5 antibody (JacksonImmuonResearch laboratories Inc. 515-175-045) diluted in 1% BSA, 1%fetal bovine serum, 0.5% TWEEN-20, 0.1 mM ZnCl₂, 1 mM MgCl₂ in PBSbuffer pH 7.4.

200 ul of reagent H were added to reservoir C of the first lateral flowcapillary device.

200 ul of reagent H were added to reservoirs B and C of the secondlateral flow capillary device.

200 ul of reagent H were added to reservoirs A, B and C of the thirdlateral flow capillary device.

200 ul of reagent H were added to reservoirs A, B and C of the fourthlateral flow capillary device. After complete draining of liquid fromreservoir C, and additional 200 ul of reagent H were added to reservoirC.

There was a linear correlation between the intensity of fluoresenceemitted by a given test line 20 b as measured by PART Immuno Reader (LRETechnology Partner GmbH) and the total volume of liquid added to therespective lateral flow capillary device, see the graph in FIG. 10.

Experiment 10 Detection of HPV 16 DNA Sequence

A lateral flow capillary device were prepared substantially as describedin Experiment 1 where capillary flow matrix 18 was nitrocellulose Prima40 (Schleicher & Schuell).

A reaction zone 20 was prepared by applying a line of spots 36 mm fromthe upstream end of the capillary flow matrix, each spot produced byapplying 1 microliter of 5 ug/mL oligonucleotide probe(5′GTTTCAGGACCCACAGGAGCGACCC (nt 106-130)) in 1.5 M NaCl and 0.15MNa-citrate, pH 7.0 solution. After drying at 37° C. for 15 minutes,capillary flow matrix 18 was irradiated with ultraviolet light for 5minutes to fix the DNA to capillary flow matrix 18.

Cellular DNA from CaSki cells was subjected to 30 PCR amplificationcycles using a first primer (5′AAGGGCGTAACCGAAATCGGT (nt 26-46)) and abiotinylated second primer (5′GTTGTTTGCAGCTCTGTGC (nt 150-168)) specificfor HPV 16 sequences. PCR was ended with a denaturation step and fastchilling to 4° C.

In reservoir A of the lateral flow capillary device was placed 50 ul ofdenaturated biotinylated PCR product, diluted 1:10 in ice chilled 0.6 MNaCl, 0.02% Ficoll 400, 0.02% gelatin, 1% PVP, 20 mM phosphate buffer pH7.5 solution.

In reservoir B of the lateral flow capillary device was placed 50 ul ofStreptavidin-Alkaline Phosphatase conjugate (Jackson ImmuonResearchlaboratories Inc. 003-050-083) diluted in 1% BSA, 0.5% TWEEN 20, 0.1 mMZnCl₂, 1 mM MgCl₂ in PBS buffer pH 7.4.

In reservoir C of the lateral flow capillary device was placed 150 ul ofreagent C, BCIP/NBT was prepared according to manufacturer instruction:stock preparation—1 tablet BCIP (Sigma-Aldrich B0274) dissolving in 1 mlDMF, 1 tablet NBT (Sigma-Aldrich N55141) dissolving in 1 ml water. Finalsolution: 33 ul BCIP, 333 ul NBT stock solutions, in 10 ml 0.1M Trisbuffer pH 9.7.

Sequential draining of reservoirs A, B, and C in accordance with theteachings of the present invention was observed. After the liquid inreservoir C completely drained away, a purple colored line at thereaction zone indicated the presence of the HPV 16 DNA sequences.

Experiment 11 Detection of HIV-1 Antibodies Using Lyophilized Conjugatein a Reaction Zone

A lateral flow capillary device was prepared substantially as describedabove in Experiment 7 except that lyophlized reagents were placed asfollows:

In the reaction zone was placed lyophlized (as described above) solutionof 1 mg/ml biotinylated synthetic gp41 and gp120 peptides diluted in 1%BSA, 1% fetal bovine serum, 0.5% TWEEN-20 in PBS buffer pH 7.4

Reservoir A was kept empty.

In reservoir B was placed a lyophilized (as described above) solution ofStreptavidin-Alkaline Phosphatase conjugate (Jackson ImmuonResearchlaboratories Inc. 003-050-083) diluted in 1% BSA, 0.5% TWEEN-20, 0.1 mMZnCl₂, 1 mM MgCl₂ in PBS buffer pH 7.4.

In reservoir C was placed a lyophilized (as described above) solution ofBCIP/NBT prepared according to the manufacturer instructions stockpreparation—1 tablet BCIP (Sigma-Aldrich B0274) dissolving in 1 ml DMF,1 tablet NBT (Sigma-Aldrich N55141) dissolving in 1 ml water. A finalsolution: 33 ul BCIP, 333 ul NBT stock solutions, in 10 ml 0.1M Trisbuffer pH 9.7.

150 ul of a serum sample were applied to capillary flow matrix 18through observation window 22, 150 ul of serum sample were added toreservoir B and 300 ul of double distilled water were added to reservoirC. Sequential draining of reservoirs B and C in accordance with theteachings of the present invention was observed. After the liquid inreservoir C completely drained away 120 ul 0.25 M sulfuric acid stopsolution was added to reservoir C.

The appearance of two colored dotted lines, one at the test line and theother at the control line, indicated the presence of antibodies for HIV1 in the serum sample.

In the experimental section above the teachings of the present inventionwere exemplified for the study of enzymatic reactions and for thedetection of specific analytes in a sample. As is clear to one skilledin the art upon perusal of the description herein, the teachings of thepresent invention are applicable to many different fields where theperformance of multistep reactions are required, including but notlimited to environmental chemistry, cell biology and biochemistry.

Methods and processes have been described herein as a series of steps inan order selected as being the easiest to understand. It must beemphasized that such order is not limiting, and any method or processdescribed herein may be implemented where the steps are performed in anyreasonable order to achieve the desired result.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, the present invention is intended to embrace all suchalternatives, modifications and variations that fall within the spiritand broad scope of the appended claims. For example, the teachings ofthe present invention have been described where a reaction takes placeat room temperature. In embodiments of the present invention, a lateralflow capillary device is maintained in warmer or colder environment, forexample a freezer, a refrigerator, or an incubator so that a reaction isperformed at a temperature that is hotter or colder than roomtemperature or to ensure that a specific desired temperature ismaintained. Embodiments in which a lateral flow capillary device ismaintained at a controlled temperature include during an entire reactionor during only part of a reaction.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. In caseof conflict, the specification herein, including definitions, willcontrol. Citation or identification of any reference in this applicationshall not be construed as an admission that such reference is availableas prior art to the present invention.

1. A lateral flow capillary device comprising: a) a unipath bibulouscapillary flow matrix having an upstream end and a downstream enddefining a flow direction; b) at least two reservoirs in fluidcommunication with said capillary flow matrix each through at least onerespective liquid receiving zone; wherein a said reservoir contacts arespective said liquid receiving zone through an opening constituting ahollow conduit having a rim pressing said matrix and wherein a portionof said capillary flow matrix between said two rims is an interfacecreation zone.
 2. The device of claim 1, wherein said pressing is suchthat liquid induced swelling of said matrix is constrained.
 3. Thedevice of claim 1, wherein said rims press said matrix when said matrixis dry.
 4. The device of claim 1, wherein said rims are pressed intosaid matrix when said matrix is dry.
 5. The device of claim 1, whereinsaid rims are substantially parallel to said flow direction.
 6. Thedevice of claim 1, wherein pressure applied by a said rim issubstantially uniform about the entire surface of said rim.
 7. Thedevice of claim 1, said matrix being substantially compressible.
 8. Thedevice of claim 1, said matrix comprising glass fibers.
 9. The device ofclaim 8, said matrix consisting essentially of glass-fibers.
 10. Thedevice of claim 1, wherein the internal surface-area volume⁻¹ of saidmatrix proximate to a said rim is higher than distant from said rim. 11.The device of claim 1, wherein opposite each said rim is disposed asupporting component supporting said matrix against said pressing. 12.The device of claim 11, wherein said matrix is suspended between saidrims and said supporting components.
 13. The device of claim 1, whereinsaid matrix is attached to a substantially impermeable backing.
 14. Thedevice of claim 13, wherein said impermeable backing contacts at leastone supporting component supporting said impermeable backing againstsaid pressing.
 15. The device of claim 13, wherein opposite each saidrim is disposed a said supporting component supporting said matrixagainst said pressing.
 16. The device of claim 15, wherein said matrixis suspended between said rims and said supporting components.
 17. Thedevice of claim 1, further comprising, downstream from at least one saidliquid receiving zone, a reaction zone comprising at least one capturingentity configured to capture a material flowing through said capillaryflow matrix.
 18. The device of claim 1, further comprising, downstreamfrom at least two said liquid receiving zones, a reaction zonecomprising at least one capturing entity configured to capture amaterial flowing through said capillary flow matrix.
 19. The device ofclaim 1, further comprising a liquid drain in fluid communication withsaid capillary flow matrix downstream from at least two of said at leasttwo reservoirs.
 20. The device of claim 1, wherein said fluidcommunication through said liquid receiving zones is non-capillary fluidcommunication. 21.-53. (canceled)